Mice homozygous for the fat mutation exhibit marked hyperpro-insulinemia and develop late onset obesity. The fat mutation was recently mapped to the gene encoding carboxypeptidase E (CpE), a processing enzyme involved in trimming C-terminal paired basic residues from prohormone-derived peptides. The mutation resulted in a loss of CpE activity that correlated with aberrant proinsulin processing. Neurotensin (NT) and melanin-concentrating hormone (MCH) are two neuropeptides that, among other central effects, inhibit food intake. Here, using RIA techniques coupled to reverse phase HPLC, we analyzed the processing products derived from the NT and MCH precursors in the brain of +/fat and fat/fat mice. Compared to control hypothalamic and brain extracts, fat/fat extracts had markedly reduced levels (>80%) of NT and neuromedin N (NN), another active pro-NT-derived peptide. In contrast, they exhibited high concentrations of biologically inactive NT-KR and NN-KR (NT and NN with a C-terminal Lys-Arg extension), two peptides that were undetectable in control extracts. MCH, which is located at the C-terminus of its precursor, was present in 2- to 3-fold higher amounts in fat/fat than in +/fat hypothalamus. Neuropeptide-Glu-Ile, another pro-MCH-derived neuropeptide separated from MCH by an Arg-Arg sequence, was present in amounts similar to those of MCH in control extracts. In contrast, neuropeptide-Glu-Ile was more than 10 times less abundant than MCH in extracts from obese mice. Our data are consistent with a deficit in CpE activity affecting the maturation of both pro-NT and pro-MCH. This suggests that abnormal neuropeptide and hormone precursor processing is a general phenomenon in fat/fat mice and supports the idea that defects in the production of neuropeptide involved in the control of feeding might lead to the development of obesity in these animals.